Fluorescence applications penetrate nearly every field of biological research and have more recently been proposed as a means for light-based computational applications by virtue of a single fluorophore's observed propensity to undergo a binary switch between fluorescent and dark states. In vitro and in vivo fluorescence measurements, as well as wide-field, scanning confocal, and Total Internal Reflection Fluorescence Microscopy (TIRF) used for whole cell and single-molecule imaging rely on high-quantum yield, stable fluorescent species such as those shown in FIG. 3.
However, the utility of such fluorophores, including organic dyes, fluorescent proteins, as well as inorganic quantum dots and nanocrystals, is limited by their intrinsic photophysical properties that lead to transient and/or permanent dark states. It is believed that these dark states arise via electronic transitions from the singlet ground and/or excited states to triplet dark states, as depicted by a simplified Jablonski diagram shown in FIG. 1. From triplet states, deleterious physical modifications or damage can occur to the dye.
For example, such processes tend to limit photon emission from the fluorophore including stochastic “blinking” events and irreversible photobleaching. Blinking and photobleaching phenomena occur in all fluorescence applications but are particularly pronounced in experiments demanding intense illumination, including confocal imaging of cells and single-molecule fluorescence methods.
In order to characterize the blinking and photobleaching behaviors adequately, individual fluorophores must be tracked as a function of time. Here, the application of modern single-molecule fluorescence methods is ideal. Single, fluorescing molecules can be easily tracked using total internal reflection (TIR) fluorescence methods where the fluorophore is spatially tethered near an optically-transparent surface and illuminated by a single-frequency laser light source, as shown, for example, in FIG. 2. In such an experimental setting, blinking and photobleaching appear as brief periods of fluorescence punctuated by long-lived non-fluorescing states, as shown, for example in FIGS. 4A-4C. Although for some applications this switching behavior may be detrimental, this characteristic may be harnessed (e.g., appropriately adjusted, modified, or even enhanced) for such applications as super-resolution imaging, computational applications, and sensor technologies.
Compounds such as Trolox, p-nitrobenzyl alcohol (NBA), β-mercaptoethanol (BME), mercaptoethylamine (MEA), n-propyl gallate, 1,4-diazabicyclo[2.2.2]octane (DABCO), and cyclooctatetraene (COT) that favorably affect dark state and photobleaching lifetimes have come into increasingly widespread use as additives in solution-based experiments. However, the use of such protective agents in solution is limited by their solubility (Trolox, COT and NBA, in particular, are highly insoluble in aqueous solutions). Examples are shown in FIG. 5. Moreover, if beneficial outcomes are required for fluorescence imaging in cells, the protective agent's membrane permeability and potential toxicity must also be considered.